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Gene Therapy and Molecular Biology Vol 13, page 1

1

Gene Ther Mol Biol Vol 13, 01-10, 2009

New trends in aptamer-based electrochemical

biosensors Review Article

Maria N. Velasco-Garcia*, Sotiris Missailidis Department of Chemistry and Analytical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes,

United Kingdom, MK7 6AA

__________________________________________________________________________________

*Correspondence: Maria N. Velasco-Garcia, Department of Chemistry and Analytical Sciences, Faculty of Science, The Open

University, Walton Hall, Milton Keynes, United Kingdom, MK7 6AA; e-mail: m.n.velasco@open.ac.uk

Sotiris Missailidis, Department of Chemistry and Analytical Sciences, Faculty of Science, The Open University, Walton Hall, Milton

Keynes, United Kingdom, MK7 6AA; e-mail: s.missailidis@open.ac.uk

Key words: Aptamer, Biosensor, Aptasensor, Electrochemical detection, SELEX

Abbreviations: Platelet-derived growth factor BB (PDGF-BB); reverse-transcription PCR (RT PCR); self-assembled monolayers

(SAMs); Systematic Evolution of Ligands by EXponential enrichment, (SELEX)

Received: 28 January 2009; Revised: 6 February 2009

Accepted: 6 February 2009; electronically published: February 2009

Summary The analytical characteristics of aptamers are comparable with those of antibodies for the development of biosensor

technology. However, aptamers offer some crucial advantages over antibodies such as selection capability for a

variety of targets, easy synthesis, improved reproducibility and stability, simple modification for immobilization to

solid supports and enhanced selectivity. This article reviews aptamer technology as well as aptamer-based assay

configurations and goes on to explore reported applications in electrochemical aptasensors.

I. Introduction Biosensor technology holds a great promise for the

healthcare market, the security sector, the food industry,

environmental and veterinary diagnostic; harnessing the

specificity and sensitivity of biological-based assays

packaged into portable and low cost devices which allow

for rapid analysis of complex samples in out-of-laboratory

environments. However the application of biosensors lags

far behind the fundamental research; the challenges facing

this basic technology are associated with sensitive

detection of specific molecules in samples, stability issues,

quality assurance, instrumentation design and cost

considerations (Velasco-Garcia and Tottram, 2003).

The main biological sensing materials used in

biosensor development are the couples enzyme/substrate

and antibody/antigen. These are limited by temperature,

sensitivity, stability, batch-to-batch variation, large size

and difficulty in production. Recent advances and

developments in the aptamer area offer a powerful

alternative approach involving the use of small RNA or

DNA molecules that bind to specific targets with very high

affinity and specificity. Aptamer receptors are a novel

entity of undeniable potential in analytical applications

and can complement or substitute antibodies or offer

applications where the later are not compatible (Tombelli

et al, 2005, 2007).

Despite the fact that development of aptasensors has

been boosted by using optical and acoustic transducers,

this review summarizes the recent developments in the

design of electrochemical aptamer-based affinity sensors.

In comparison with other detection systems, the

electrochemical detection combines a high sensitivity,

direct electronic signal production, fast response,

robustness, low cost, the possibility of miniaturization and

simultaneous multianalyte detection.

II. Aptamers As aptamers approach 20 years since they were

originally described (Ellington and Szostak, 1990; Tuerk

and Gold, 1990), they are currently receiving a wider

recognition in the literature as research reagents,

inhibitors, imaging or diagnostic agents (Luzi et al, 2003;

Hamula et al, 2006). Aptamers are short, single stranded

oligonucleotides, which inherently adopt stable three

dimensional sequence-dependent structures. This intrinsic

property makes them efficient binding molecules, capable

of binding to an array of molecular targets ranging from

small ions and organic molecules to large glycoproteins

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors

2

and mucins (Ferreira et al, 2006). Aptamers are a novel

and particularly interesting targeting modality, with the

ability to bind to a variety of targets including proteins,

peptides, enzymes, antibodies and cell surface receptors,

as well as small molecules ranging from glucose and

caffeine, to steroids to TNT. Aptamers are single stranded

oligonucleotides that vary in size between 25-90 bases

long and adopt complex secondary and tertiary structures,

which facilitate specific interactions with other molecules.

They are derived from vast combinatorial libraries through

selective targeting and competitive binding. There are two

different configurations of aptamers: (i) linear and (ii)

molecular beacon. Aptamers with a linear configuration

maintain in certain physicochemical conditions a typical 3-

D conformation with specific binding sites for the target

molecule. On the other hand aptamers with a molecular

beacon configuration initially form a loop that changes

conformation following binding to the analyte of interest.

Aptamers offer unique benefits compared to other

targeting agents; not only they bind specific ligands with

high affinity and selectivity, but aptamers can be easily

selected using in vitro techniques and are chemically

synthesized, overcoming the use of animal for their

production. In comparison to antibodies, aptamers are

purified to a very high degree of purity, which eliminates

the batch-to-batch variation found in antibodies. Aptamers

have higher temperature stability (stable at room

temperature) and because of their small size, denser

receptor layers could be generated. The animal-free

production of aptamers is especially advantageous in cases

where the immune response can fail when the target

molecule (e.g. a protein) has a structure similar to

endogenous proteins or when the antigen consists of toxic

or non-immunogenic compounds. Aptamers are relatively

stable under a wide range of buffer conditions and

resistant to chemical degradation, although, due to their

DNA or RNA constitution, they are sensitive to hydrolytic

digestion by nucleases. Aptamers have been modified into

nuclease-resistant moieties by modification of the ribose

ring at the 2’-position or by the specific modification of

the pyrimidine nucleotide (Pieken et al, 1991; Heidenreich

and Eckstein, 1992; Kusser, 2000). It is also possible to

chemically modify aptamers to facilitate covalent

conjugation to reporters and nanoparticles with 5’ or 3’

amino, biotin or thiol groups. These characteristics make

them extremely attractive as alternatives to antibodies and

peptides for use in assays, or as diagnostic agents.

A. The SELEX process Aptamers are typically isolated from combinatorial

libraries by a process of in vitro evolution, termed SELEX

(Systematic Evolution of Ligands by EXponential

enrichment). This procedure is an in vitro evolutionary

selection process that allows the isolation of aptamer(s),

with unique binding properties, from a large library of

oligonucleotides through iterative cycles of (i) interaction

of a large library of aptamers with the target molecule, (ii)

separation of bound from unbound aptamer species, (iii)

elution of bound aptamers and (iv) PCR amplification of

the binding aptamers for further selection rounds (Figure

1 for an example of the process).

Library is incubated with target

under desired selection conditions

Unbound or non-specifically

bound species are washed

away and discarded

Unbound or non-specifically

bound species are washed

away and discarded

Aptamers are interacting

with and separated from

target using various separation

techniques, including affinity

chromatography columns,

magnetic bids or membranes

Desalted aptamers

are lyophilised and

PCR reagents added

for amplification

Amplified aptamers

are used in successive

selection rounds

Aptamers from the

final selection round

are double stranded

and cloned in E. coli

Clones are sequenced

for identification of

high binding aptamer

species to be used

subsequently for

further development

Bound aptamers are

eluted from their

target using high

salt or chaotropic

agents and desalted

for further PCR

amplification steps

Positive clones are analysedPositive clones are analysed

Figure 1. The SELEX process.

Gene Therapy and Molecular Biology Vol 13, page 3

3

An aptamer library usually consists of a variable

region (20-40 nucleotides) flanked by known primer

sequences on either end for the amplification during the

SELEX procedure. The variable region makes up to 1015

different sequences which, combined with the innate

ability of oligonucleotides to form stable sequence-

dependent structures, provide an array of molecular shapes

available for the selection process (Khan and Missailidis,

2008). In the selection steps, the library is incubated with

the immobilised target. Unbound or weak-binding species

are removed and bound aptamers are eluted using high

salt, temperature, chaotropic agents or other such

conditions that would affect molecular structure or disrupt

molecular interactions. Eluted aptamers are subsequently

amplified by PCR (DNA) or reverse-transcription PCR

(RT PCR) using primers complementary to the flanking

sequences in the aptamer library. The enriched pool of

binding species forms the pool for the next round of

selection. Repeated selection and amplification steps allow

identification of the highest binding species, through

competitive binding. The selection and amplification step

constitutes one round or cycle in a typical SELEX

procedure, with anything between 1 and 15 cycles often

described in the literature. Counter- or negative selection

steps can ensure that the finally selected aptamers are very

specific for their target and do not interact with

homologous proteins or chemically closely-related

molecular targets (Missailidis, 2008).

Selected aptamers are subsequently cloned and

sequenced to identify the sequence of the binding species

and their interactions are usually characterised by a variety

of analytical methodologies, prior to move into the various

applications they were originally destined for. Selected

aptamer can be easily produced by solid phase synthesis

and appropriate modifications can be introduced at this

stage to confer additional properties to the selected

aptamers, such as nuclease resistance (Figure 2), cross-

linking ability or improved pharmacokinetic properties.

Although SELEX has been the initial methodology

associated with aptamer selection and has remained a

robust and powerful technique, which has been adapted to

various systems and targets, a number of other

methodologies have also emerged for the selection of

aptamers. Such “non-SELEX” based methods for the

selection of aptamers include capillary electrophoresis

methodologies (Berezovski et al, 2005; Drabovich et al,

2005), isolation of aptamers with predefined kinetic and

thermodynamic properties of their interaction with the

target, without the need for amplification, allowing the use

of libraries which are difficult or cannot be amplified, or

computational methods, which are particularly important

in selecting aptamers with inhibitory activities or

sequences that undergo ligand dependent conformational

changes (Ikebukuro et al, 2005).

The SELEX procedure and subsequent technologies for

aptamer selection have offered the tools for the designing

of aptamers that have found a range of diagnostic

applications (Khan and Missailidis, 2008). Such

applications include Photo-SELEX (www.somalogic.com)

and SELEX NADIR (Winters-Hilt, 2006) using optical

probe reporting or nanopore reporting mechanisms

respectively, aptamer microarrays (Cho et al, 2005),

currently in the market by LC Sciences

(www.lcsciences.com), fluorescent aptamers in chips and

microspheres (Potyrailo et al, 1998; Kirby et al, 2004),

fluorescent sensors for small molecule recognition

(Yamana et al, 2003; Ozaki et al, 2006), quantum dots

(Levy et al, 2005; Choi et al, 2006; Ivanovic et al, 2007;

Liu et al, 2007), colorimetric detection (Liu and Lu, 2004;

Cho et al, 2006; Liu and Lu, 2006), electrochemical

detection (Xiao et al, 2005; Mir et al, 2006; Lai et al,

2007; Papamichael et al, 2007) and piezoelectric quartz

crystal sensors (Bini et al, 2007).

The above methods, fluorescent, electrochemical and

colorimetric detection, have also been used in molecular

switch type sensors or modular sensor assemblies, where

the aptamers usually change conformation upon binding to

either emit a fluorescent signal based on an aptamer

beacon on sensor, or through non-covalent interaction with

the fluorescent label, triggering an electrochemical sensor

or leading to change of colour (Frauendorf and Jaschke,

2001; Stojanovic et al, 2001; Stojanovic and Landry,

2002; Stojanovic and Kolpashchikov, 2004; Baker et al,

2006; Zuo et al, 2007), with particular sensitivities in the

recognition of small analytes.

Aptamers have also been used in enzymatic sensing,

without the use of any label or signal related directly to the

aptamer. These applications remain based on changes in

the conformation of bifunctional aptamers that recognise

the target ligand and an enzyme or ribosome. The binding

of the aptamer to the ligand results in conformational

changes that affect enzymatic activity or protein

expression, and it is the later that is subsequently

measured (Ogawa and Maeda, 2007; Yoshida et al, 2006;

Yoshida et al, 2006) or utilises an enzyme to ligate

N

NN

N

NH2

O

HO

HH

HH

PO

O

O

O-

N

NH2

ON

O

NH2 or FOH

HH

HH

Figure 2. An amino or fluoro modification at the 2’ position of

the sugar can confer the oligonucleotide aptamer stability against

nuclease degradation. An alternative to using modifications at the

2’ of the sugar (whether at the 3’ or 5’ end of the aptamer, or

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors

4

both) for nuclease resistance is to use a flipped base added to the

end of the aptamer.

proximally bound aptamers to large protein targets and

allow their subsequent PCR amplification (Fredriksson et

al, 2002).

III. Aptamer immobilisation Aptamers can certainly be used as molecular

recognition elements in affinity sensing. The small size of

aptamers provides advantages over antibodies: (i) a greater

surface density of receptors and (ii) multiple binding to

target molecules for sandwich assays.

The method of immobilization of aptamers to a solid

support affects the sensitivity of the aptamer to the target

molecule. Thus, the selected method should maintain the

binding affinity and selectivity that the aptamers display in

solution (Balamurugan et al, 2008).

Aptamers can be attached to the solid support at

either the 5’-end or the 3’ end. Both positions have been

reported as being used for aptasensor development.

However, there are very few studies looking at the effect

of the two types of end attachment. Recent work suggests

that it depends on the particular aptamer (Cho et al, 2006),

although for biological targeting it may be that the 3’ end

is more suitable, since the 3’ end is the primary target for

exonucleases, and thus its coupling to the solid support

would simultaneously confer resistance to nucleases.

Gold is used for many electrochemical

measurements. Direct attachment of aptamers to gold

surfaces could be achieved by using a thiol-alkane linked

to the aptamer sequence. The gold surface could also be

functionalized and the type of chemistry selected is

dependent on what type of terminal functional group is

linked to the aptamer (amine, thiol or biotin termini;

Figure 3).

Gold surfaces functionalized with self-assembled

monolayers (SAMs) can address the nonspecific

adsorption of aptamer to the surface, which is a particular

problem for long oligonucleotides with larger numbers of

amine groups. Avidin-biotin technology has also been

exploited for aptamer immobilization. Strepavidin can be

physically adsorbed or covalently immobilized onto the

support and the method mainly requires incubation of the

biotin-tethered aptamer with the modified substrate.

Studies of the anti-thrombin aptamer revealed this

biocoating method gives best results regarding sensitivity

compared to other immobilization strategies (Hianik et al,

2007).

IV. Electrochemical assays In principle, aptamers can be selected for any given

target, ranging from small molecules to large proteins and

even cells. When aptamers bind small molecular targets,

these get incorporated into the nucleic acid structure,

buried within the binding pockets of aptamer structures.

On the other hand, large molecules (e.g. proteins) are

structurally more complicated, allowing aptamer

interactions at various sites via hydrogen bonding,

electrostatic interactions and shape complementarity. The

use of aptamers as bio-recognition elements for small

molecules has not been reported as extensively as for

protein targets.

O

PO O

O-

CnHS

Aptamer

O

PO O

O-

CnBiotin

Aptamer

O

PO O

O-

CnH2N

Aptamer

Figure 3. Standard nucleic acid modifications used for aptamer

immobilisation. Most of the common modifications are linked

via the phosphate group of the oligonucleotide aptamer. Various

lengths carbon chains are used that can offer higher or lower

flexibility.

Mainly two different assay configurations have been

reported to transduce these target-binding aptamer events:

(i) single-site binding and (ii) dual-site binding (Song et al,

2008). Small molecules are often assayed using the single-

site binding configuration. Protein targets can be assayed

via both single-site and dual-site binding. The dual-site

binding assay is commonly known as the sandwich assay.

Normally, the target molecule is sandwiched between a

pair of aptamers that bind to different regions of the large

molecule. One aptamer is immobilized on a suitable solid

support to capture the target while the other aptamer for

detection is conjugated to a catalytic label. Enzymes,

inorganic or organic catalysts or nanoparticles are often

used for electrochemical detection. In some cases, when

there is only one aptamer for the molecule of interest,

antibodies have been reported to be used instead of the

second aptamer (Ferreira et al, 2008). If the target protein

contains two identical binding sites, the selection of a

single aptamer still allows the development of a sandwich

assay.

Displacement assays have been also proposed to

overcome the more challenging detection of small

molecules. Affinity interactions between aptamers and

small ligands are weaker than interaction with large

molecules (with dissociation constants in the µM range, in

comparison with constants for large molecules that are in

the pM-nM range). The presence of the small target could

Gene Therapy and Molecular Biology Vol 13, page 5

5

induce the separation of two strands of a duplex nucleic

acid (one strand being the aptamer immobilised to a solid

support). Another strategy could rely on the displacement

of the aptamer from its complex with the immobilised

target molecule when the molecule is present in solution

(De-los-Santos-Alvarez et al, 2008).

Induced-fit conformational changes of the aptamer

after binding to the target molecule can also be used to

monitor a bio-recognition event by tagging the aptamer

(Figure 4). The use of labels requires precise knowledge

of the aptamer folding mechanism after binding to the

target and the binding sites. In the case of a redox active

marker, the accessibility of the label to the conducting

support is associated with the tertiary structure of the

aptamer before and after the binding event. However, for

small molecules, this strategy is not always viable,

because the aptamer 3D structure could only be slightly

perturbed after the ligand interaction.

Redox-active reporting labels could not be covalently

tethered to aptamers. Methylene blue has been intercalated

into the double-stranded DNA domain of a hairpin

configuration aptamer. The binding of the target with the

aptamer opens the hairpin structure and releases the

intercalated methylene blue. As a result, the amperometric

response decreased with the addition of the analyte. This

approach is known as “label-free” method (Figure 5).

Figure 4. Assays based on induced-fit conformational changes of aptamers.

Figure 5. Label-free electrochemical assays based on: (A) methylene blue intercalated into the DNA aptamer and (B) cationic redox-

active reporting units bound to DNA aptamer phosphate backbone.

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors

6

Related approaches use cationic redox-active reporting

units bound to the electrode via electrostatic interactions

with the DNA aptamer phosphate backbone. The binding

of the target molecule with the aptamer blocked the

binding of the cationic reporting units and the

electrochemical response decreased. The main

disadvantage of these latter approaches is a negative

detection signal.

Recently, nanomaterials are also providing novel

electrochemical sensing approaches. Single-walled carbon

nanotube field-effect transistor sensors were developed to

monitor aptamer-protein binding studies. Aptamers are

well suited for FET sensing due to their small size (1-2

nm) and recognition occurs inside the electrical double-

layer associated with the gate (within the Debye length).

The single-walled carbon nanotubes were assembled

between source and drain electrodes and the aptamers

were immobilized to these nanomaterials. In this label-free

approach, the binding of the target molecule to the

aptamers altered conductance through the device. The ease

of miniaturization of these sensing devices opens up the

feasibility of high-throughput assays in microarrays.

Nanoparticles have also been reported as catalytic

labels, instead of enzymes, and carriers for ultrasensitive

electrochemical detection; because one nanoparticle

contains a large number of aptamers, the target binding

process is amplified.

Impedance spectroscopy has been the most

frequently used electrochemical method in the

development of electrochemical aptasensors and has

shown excellent sensitivity, achieving limit of detection of

fM. However, despite the fact that the analytical technique

is simple to perform, the data fitting remains a bit

complicated. Easier data processing and faster response

could be achieved with chonoamperometry, but the limit

of detection will be higher and in the nM range.

IV. Applications of electrochemical

aptasensors Aptamer publications have now appeared in the

literature using most of the electrochemical transducers.

The majority of aptamer work on electrochemical sensors

is focused on amperometric transducers, but there have

been references on aptamers used in impedimetric, FET

and recently potentiometric sensors. Furthermore, a lot of

the work on the aptamers in electrochemical sensors has

been on the model protein, thrombin, which is one of the

best characterised complexes in the aptamer literature.

These have provided proof of principle concepts as to how

aptamers could be developed in novel sensors. However, a

number of other systems have also now been described,

which will be presented in this review.

A. Electrochemical aptasensors for the model

protein The thrombin-binding aptamer (15-mer, 5’-

GGTTGGTGTGGTTGG-3’) was the first one selected in

1992 by Block and colleagues and its structure has been

well characterized and studied. The folded structure in

solution is composed of two guanine quartets connected

by two T-T loops spanning the narrow grooves at one end

and a T-G-T loop spanning a wide groove at the other end

(known as the G-quartet structure). This anti-thrombin

aptamer has been extensively used as the model

oligonucleotide by many researchers to demonstrate the

wide applicability of aptamers as bio-recognition elements

in biosensors.

In the literature, many different electrochemical

aptasensors for thrombin detection have been reported.

The most straightforward configuration is based on the

immobilization of a thiol terminated aptamer on a gold

electrode. The aptamer-thrombin interaction is transduced

by the electrochemical quantification of p-nitroaniline

produced by the thrombin’s enzymatic reaction. Thrombin

has two electropositive exosites both capable of binding

the aptamer, allowing the development of an

electrochemical sensor system in a sandwich manner. The

thiolated aptamer was immobilized on a gold electrode

and, after incubation with the thrombin, a second

incubation step with an HRP labelled aptamer took place.

Electrochemical detection of HRP was performed using

H2O2 and a diffusional osmium based mediator. A similar

aptasensor system in sandwich manner for thrombin was

developed based on the aptamer for detection, labelled

with pyrroquinoline quinine glucose dehydrogenase, and

the electric current generated from glucose addition after

the formation of the complex on a gold electrode

(Ikebukuro et al, 2005). Another strategy for the thrombin

sensing is the direct immobilization of the protein on the

electrode surface. After the incubation with biotin-labelled

aptamer and then with streptavidin-HRP, the

electrochemical detection is performed using H2O2 and a

diffusional osmium-based mediator. The latter approach

achieved the lower limit of detection, 3.5 nM (Mir et al,

2006).

Mir and colleagues also developed in 2008 a

chronoamperometric beacon biosensor based on a

ferrocene-labelled thiol-aptamer. The aptamer adopts a 3-

D conformational change after binding the thrombin,

allowing the ferrocene label to approach to the gold

electrode. The interaction is detected via a

microperoxidase mediated electron transfer between the

label and the electrode surface. The system was

demonstrated with impedance spectroscopy and

chronoamperometry measurements, achieving a limit of

detection of 30 fM with the impedance spectroscopy (Mir

et al, 2008).

Methylene blue has also been used as an

electrochemical marker. The beacon aptamer surface was

prepared following formation of 11-mercaptoundecanoic

acid self-assembled monolayer on gold electrode.

Methylene blue was intercalated on the aptamer by the

interaction with two guanine bases. Binding of the

thrombin is correlated with the decrease in electrical current intensity in voltammetry. The estimated detection

limit of the target thrombin was 11 nM (Bang et al, 2005).

The modification of antibodies is difficult, costly and

time consuming; however researchers have been using

conventional polyclonal antibodies as a capturing probe

and labelled-aptamers as the detection probe in new

sandwich approaches for protein detection. Kang and

Gene Therapy and Molecular Biology Vol 13, page 7

7

colleagues reported in 2008 a modified electrochemical

sandwich model for thrombin, based on capturing

antibody immobilized onto glassy carbon electrodes with

nanogold-chitosan composite film and Methylene blue

labelled aptamer as the electrochemical detection probe.

Lu and colleagues described in 2008 an

electrochemical aptasensor for thrombin that is not based

on the target binding-induced conformational change of

aptamers. The thrombin-binding aptamer is first assembled

onto a gold electrode and then hybridized with a ferrocene

labelled short aptamer-complementary DNA

oligonucleotide. The binding of the thrombin to the

aptamer destroys the double-stranded DNA

oligonucleotide and leads to the dissociation of the label

short complementary DNA oligonucleotide from the

electrode surface, resulting in a decrease in the differential

pulse voltammetry responses at the electrode (Lu et al,

2008). This strategy is based on the stronger binding

affinity of the aptamers towards their targets rather than to

the short aptamer-complementary DNA oligonucleotide

labelled with electroactive moieties.

The majority of the work performed on aptamer-

based electrochemical biosensors is based on aptamers

labelled using redox compounds, such as methylene blue,

and catalysts such as horseradish peroxidase. However,

nanoparticle-based materials offer excellent prospects for

a new signal amplification strategy for ultrasensitive

electrochemical aptasensing. Platinum nanoparticles have

been reported as catalytic labels when linked to a thiolated

aptamer. The nanoparticles catalysed the electrochemical

reduction of H2O2 and the resulting current enabled the

amplified detection of thrombin sandwiched between the

aptamer on the electrode surface and the aptamer labelled

with the nanoparticles (Polsky et al, 2006). Gold

nanoparticles offer several advantages such as electrical

conductivity, biocompatibility, ease of self-assembly

through a thiol group, increase electrode surface area and

amount of immobilized capturing probe. Gold

nanoparticles have been used as an electrochemical

sensing platform for direct detection of thrombine. The

aptamer was immobilised on a screen-printed electrode

modified with gold-nanoparticles by avidin-biotin

technology. The gold-nanoparticles surface status is

evaluated by the Au/Au oxide film formation with cyclic

and stripping voltammetry. Gold nanoparticles signal

changed with the deposition of biolayers due to

differences in electron transfer efficacy and availability of

buffer oxygen. Aptamers prefer to adopt the G-quarter

structure when binding with thrombin and the

conformational changes made double strand DNA zones

appear and facilitated the electron transfer from solution to

the electrode surface, based on the double stranded DNA’s

ability to transport charge along the nucleotide stacking

(Suprun et al, 2008). The detection limit of this novel

approach is in the nM range. However, the aptasensor

measured directly binding events and opened 4 orders of

magnitude the operating range of protein concentration.

Assays coupling aptamers with magnetic beads for

the aptamer or target immobilisation before the

electrochemical transduction have also been proposed

(Centi et al, 2008). The use of magnetic beads improved

the assay kinetics due to the beads being in suspension and

also minimized matrix effect because of better washing

and separation steps.

An ultrasensitive electrochemical aptasensor for

thrombin in a sandwich format of magnetic nanoparicle-

immobilized aptamer, thrombin and gold nanoparticle-

labelled aptamer was reported by Zheng and colleagues in

2007. The magnetic nanoparticle-immobilized aptamer

was used for capturing and separating the target protein.

The gold nanoparticle-labelled aptamer offered the

electrochemical signal transduction. The signal was

amplified by forming a network like thiocyanuric acid/

gold nanoparticles to cap more nanoparticles per assay,

lowering the detection limit to the aM range

B. Other targets Aptamer have been selected against a wide range of

targets with typical binding affinities in the nanomolar to

picomolar range. Recently, electrochemical aptasensors

have been reported to detect proteins, hormones and drugs.

Papamichael and colleagues described in 2007 a

disposable electrochemical aptasensor for

Immunoglobulin E, a key marker of atopic diseases (such

as asthma, dermatitis and pollenosis). The sensor

incorporates a competitive format for IgE detection using

a biotinylated form of the aptamer. A standard, indirect

method was used where competition between surface-

bound IgE and IgE in solution proceeded for the aptamer.

The electrochemical detection is achieved by the use of an

extravidin-alkaline phosphatase label. After careful

optimization of conditions (buffer pH, ionic strength,

additional ions and proteins), the aptasensor was

performing at levels suitable for human testing (>300ng

ml-1).

Platelet-derived growth factor BB (PDGF-BB) is one

important cytokine involved in neural inflammation and

was selected as target for the development of an

electrochemical aptasensor based on capacitance change

induced by aptamer-protein specific binding, measured by

non-faradic impedance spectroscopy. The biosensor

detection limit was 40 nM. Electrochemical impedance

spectroscopy is a very attractive method for in vivo

diagnostics, due to its high sensitivity and label free

characteristics (Liao and Cui, 2007). A similar

electrochemical detection was also reported to a

tuberculosis-related cytokine, the interferon-!. The

aptamer-based electrochemical impedance biosensor

successfully detected interferon-! to a level of 100 fM

with an RNA aptamer and 1 pM with a DNA aptamer

probe (Min et al, 2008).

Electrochemical aptasensors for 17-" estradiol have

also been reported. The selected biotinylated DNA

aptamer was immobilized on a streptavidin-modified gold

electrode. The chemical binding of the hormone to the

aptamer was monitored by cyclic and square wave

voltammetry. When the 17-" estradiol interacted with the

aptamer, the current decreased due to the interference of

the bound target molecule with the electron flow produced

by a redox reaction between ferrocyanide (the mediator)

and ferricyanide. The linear range of this aptasensing

device was 1-0.01 nM of 17-" estradiol (Kim et al, 2007).

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors

8

Cocaine has been detected by an electrochemical

aptasensor incorporating gold nanoparticles onto the

surface of a gold electrode. The thiol-derivative aptamer

was self-assembled onto the gold nanoparticles. The

aptamer was also functionalized at the other termini of the

strand with a redox-active ferrocene moiety. The cocaine

binding to the aptamer induces the conformational change

of the aptamer, bringing the redox tag in close proximity

to the electrode, leading to an increase in the current (Li et

al, 2008). Methylene blue tagged aptamer has been also

explored for the detection of cocaine (Baker et al, 2006).

A novel adenosine aptasensor was reported based on

the structure change of an aptamer probe immobilized on a

gold electrode. After the binding aptamer-target

nucleoside, a higher surface charge density and an

increasing steric hindrance were obtained that reduce the

diffusion of [Fe(CN)6]3-/[Fe(CN)6]

4- towards the electrode

surface, resulting in a decrease of the current. The

biosensing surface was easily regenerated and the

aptasensor limit of detection was 10 nM (Zheng et al,

2008).

C. Aptasensor arrays Some of the aptamer-based biosensor technology

described in this review could be transferred from single-

analyte devices to electrochemical methods offering the

possibility of simultaneous measurements of a panel of

targets. Wang reviewed the use of metal nanoparticles as

tracers for the analysis of nucleic acid hybridization.

Magnetic nanoparticles were linked to different probe

DNAs and incubated with samples containing different

DNA targets. Semiconductor quantum dots were

functionalized each with different nucleic acids

complementary to the free chain of the target DNA. After

dissolution of the metal nanoparticles, the identification of

the metal ions by stripping voltammetry enabled the

analysis of the different DNA targets (Wang, 2003).

Thrombin and lysozyme were detected in parallel

using a competitive assay in which thrombin and

lysozyme were modified with different semiconductor

quantum dots (Hansen et al, 2006). Specific aptamers were

immobilized on a gold electrode and bound to the

respective labelled protein. In the presence of unlabelled

protein in the sample, the quantum-dot functionalized

protein is displaced from the electrode into solution. The

dissolution of the remaining metal ions on the surface and

the electrochemical detection of the released ions enabled

the quantitative detection of the proteins.

IV. Conclusions Aptamers have been widely used in a variety of

diagnostic and sensor applications, offering a variety of

possibilities for aptamer-based sensors in early disease

diagnosis and prognosis, substance control, environmental

measurements or national security applications on

measurements of explosives or potential infectious agents.

Yet, despite the advances and the huge body of literature

documenting the success of the technology, the

commercial application of aptamers in the field of

diagnostics remains relatively undeveloped, not least due

to the exclusive IP portfolio, and the fact that there is a

vast antibody-based diagnostic market and a certain degree

of hesitation to move to a new type of product, unless

aptamers offer verifiably significant improvements on

current technologies that warrant substitution of antibodies

in some current assay formats. In this review, different

types of electrochemical aptamer-based biosensors have

been discussed. Although the optical and mass-sensitive aptasensors have been the most commonly described in

the literature, electrochemical transducers have enormous

potential and offer simple, rapid, cost-effective and easy to

miniaturize sensing in many diagnostic fields. Emerging

nanomaterials have also brought new possibilities for

developing novel ultrasensitive electrochemical

aptasensors.

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Maria N. Velasco-Garcia and Sotiris Missailidis